A TLR9-dependent checkpoint governs B cell responses to DNA-containing antigens
Vishal J. Sindhava, Michael A. Oropallo, Krishna Moody, Martin Naradikian, Lauren E. Higdon, Lin Zhou, Arpita Myles, Nathaniel Green, Kerstin Nündel, William Stohl, Amanda M. Schmidt, Wei Cao, Stephanie Dorta-Estremera, Taku Kambayashi, Ann Marshak-Rothstein, Michael P. Cancro
Vishal J. Sindhava, Michael A. Oropallo, Krishna Moody, Martin Naradikian, Lauren E. Higdon, Lin Zhou, Arpita Myles, Nathaniel Green, Kerstin Nündel, William Stohl, Amanda M. Schmidt, Wei Cao, Stephanie Dorta-Estremera, Taku Kambayashi, Ann Marshak-Rothstein, Michael P. Cancro
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Research Article Immunology

A TLR9-dependent checkpoint governs B cell responses to DNA-containing antigens

Abstract

Mature B cell pools retain a substantial proportion of polyreactive and self-reactive clonotypes, suggesting that activation checkpoints exist to reduce the initiation of autoreactive B cell responses. Here, we have described a relationship among the B cell receptor (BCR), TLR9, and cytokine signals that regulate B cell responses to DNA-containing antigens. In both mouse and human B cells, BCR ligands that deliver a TLR9 agonist induce an initial proliferative burst that is followed by apoptotic death. The latter mechanism involves p38-dependent G1 cell-cycle arrest and subsequent intrinsic mitochondrial apoptosis and is shared by all preimmune murine B cell subsets and CD27– human B cells. Survival or costimulatory signals rescue B cells from this fate, but the outcome varies depending on the signals involved. B lymphocyte stimulator (BLyS) engenders survival and antibody secretion, whereas CD40 costimulation with IL-21 or IFN-γ promotes a T-bet+ B cell phenotype. Finally, in vivo immunization studies revealed that when protein antigens are conjugated with DNA, the humoral immune response is blunted and acquires features associated with T-bet+ B cell differentiation. We propose that this mechanism integrating BCR, TLR9, and cytokine signals provides a peripheral checkpoint for DNA-containing antigens that, if circumvented by survival and differentiative cues, yields B cells with the autoimmune-associated T-bet+ phenotype.

Authors

Vishal J. Sindhava, Michael A. Oropallo, Krishna Moody, Martin Naradikian, Lauren E. Higdon, Lin Zhou, Arpita Myles, Nathaniel Green, Kerstin Nündel, William Stohl, Amanda M. Schmidt, Wei Cao, Stephanie Dorta-Estremera, Taku Kambayashi, Ann Marshak-Rothstein, Michael P. Cancro

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Figure 5

Naive human B cells undergo post-proliferation apoptosis following stimulation with BCR-internalized TLR9 ligands.

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Naive human B cells undergo post-proliferation apoptosis following stimu...
(A) Representative FACS plots showing proliferation and survival in human CD27–CD19+ PBMCs cultured for 108 hours with no stimulation, ODN 2006 plus F(ab′)2 fragments of anti-IgM, hSTIC9, hSTIC9 plus BLyS, or hSTIC9 plus SB203580. Dead cells were stained by TO-PRO-3, while CFSE dilution indicates proliferation. (B) Percentage of live divided cells from multiple donors treated as in A. Each symbol represents a single donor. Black symbols (n = 3) indicate cells that were cultured with ODN 2006 plus F(ab′)2 fragments of anti-IgM and hSTIC9, while white symbols (n = 4) indicate cells that were additionally cultured with hSTIC9 plus BLyS or hSTIC9 plus the p38 inhibitor SB203580. (C) Human CD27–CD19+ PBMCs were cultured with STIC9 plus p38 inhibitor (10 μM) or STIC9 plus anti-CD40, with or without IFN-γ, for 108 hours. At the end of the culture, cells were stained for T-bet. Plots show T-bet expression in live cells. Numbers inside the plot indicate Δ MFI (experimental minus isotype control). n = 3 for all data, and results are representative of at least 3 independent experiments. **P < 0.005, by 2-tailed Student’s t test.